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British Food Journal
Nutritional and phytochemical content of Swiss chard from Montenegro, under
different fertilization and irrigation treatments
Ljubica Ivanović, Ivana Milašević, Ana Topalović, Dijana Ðurović, Boban Mugoša, Mirko Knežević,
Miroslav Vrvić,
Article information:
To cite this document:
Ljubica Ivanović, Ivana Milašević, Ana Topalović, Dijana Ðurović, Boban Mugoša, Mirko Knežević,
Miroslav Vrvić, (2018) "Nutritional and phytochemical content of Swiss chard from Montenegro,
under different fertilization and irrigation treatments", British Food Journal, https://doi.org/10.1108/
BFJ-03-2018-0142
Permanent link to this document:
https://doi.org/10.1108/BFJ-03-2018-0142
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Nutritional and phytochemical
content of Swiss chard
from Montenegro, under
different fertilization and
irrigation treatments
Ljubica Ivanovićand Ivana Milašević
Institute of Public Health of Montenegro,
Podgorica, Montenegro
Ana Topalović
Center for Soil Research and Melioration, Biotechnical Faculty,
University of Montenegro, Podgorica, Montenegro
Dijana Ðurovićand Boban Mugoša
Institute of Public Health of Montenegro,
Podgorica, Montenegro
Mirko Knežević
Center for Soil Research and Melioration,
Biotechnical Faculty, University of Montenegro,
Podgorica, Montenegro, and
Miroslav Vrvić
Faculty of Chemistry,
University of Belgrade, Belgrade, Serbia
Abstract
Purpose –The purpose of this paper is to evaluate both nutrient and phytochemical content of Swiss
chard grown under different fertilization and irrigation treatments and the effect of these treatments on the
tested parameters.
Design/methodology/approach –Samples of fresh Swiss chard were collected from the experimental
field of Ljeskopolje, Montenegro, where chard was grown under different fertilization and irrigation
treatments. Swiss chard samples were analyzed for nutritional and antioxidant parameters.
Findings –In this study, the authors found that 100 g of Swiss chard is a good source of total chlorophyll
(47.13 mg), carotenoids (9.85 mg), minerals as well as vitamin C (26.88 mg) expressed as mean values.
Total phenol and flavonoid compounds content were (138.59 µg gallic acid equivalent (GAE) and
11.91 µg catechin equivalent (CAE) per mg of water extract, respectively), also expressed as mean
values. The total antioxidant capacity (IC50 values) determined by 1,1-diphenyl-2-picrylhydrazyl
assay ranged from 2.93 to 4.44 mg/mL of aquatic water extract. Different fertilization regimes affected
the following parameters: phosphorous, protein content, chlorophyll a, chlorophyll b and vitamin C
(po0.05), while different irrigation regimes did not have any effect on the tested parameters ( pW0.05),
while interaction effect between fertilization and irrigation was found only for sodium and
copper ( po0.05).
Originality/value –Swiss chard produced in Montenegro on a sandy clay loam soil with acid reaction
contains appreciable amount of minerals, crude fibers, vitamin C, chlorophylls, carotenoids and polyphenols.
The nutrient and phytochemical content of chard is equal or superior to other green leafy vegetables which
British Food Journal
© Emerald Publishing Limited
0007-070X
DOI 10.1108/BFJ-03-2018-0142
Received 6 March 2018
Revised 21 June 2018
Accepted 29 June 2018
The current issue and full text archive of this journal is available on Emerald Insight at:
www.emeraldinsight.com/0007-070X.htm
This work has been supported by the Ministry of Science of Montenegro and the HERIC project
through the BIO-ICT Centre of Excellence (Contract No. 01-1001).
Swiss
chard from
Montenegro
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are considered as functional food. It was identified as a potentially rich source of essential nutrients and
phytochemical compounds. The promotion of higher consumption and production of Swiss chard may
represent a natural and sustainable alternative for improving human health.
Keywords Irrigation, Nutrients, Fertilization, Phytochemicals, Swiss chard
Paper type Research paper
1. Introduction
Swiss chard (Beta vulgaris ssp. cicla L) is a dark green leafy vegetable (GLV ) available
throughout the year. Swiss chard could be planted in mid spring and again in late summer,
which indicates the possibility of harvesting during a long period (Kolota et al., 2010). Chard
is a commonly used food crop which is found along the shores of the Mediterranean,
frequently used all over the world due to its nutritional value and delicious taste. Chard is a
desirable food crop because it adapts to environments with elevated saline concentrations,
and it can grow in soils with scarce availability of water (Kolota et al., 2010; Ninfali and
Angelino, 2013). Furthermore, Swiss chard is tolerant to conditions of low light and both
cold and hot weather (Kolota et al., 2010).
GLVs are exceptionally low in energy but also relatively high in micronutrients and
phytochemicals, which recommend GLVs for consumption in everyday diet (van Jaarsveld
et al., 2014). Certain epidemiological studies promoted consumption of GLVs because these
vegetables were found to protect against numerous chronic diseases caused by free radical
activity (Slavin and Lloyd, 2012). Swiss chard, as one of the GLVs, is rich in phytopigments
such as chlorophyll and carotenoids. Phytopigments improve immune, detoxication and
antioxidant systems of the human body, thus indirectly helping the prevention of disease
(Fiedor and Burda, 2014; Ferruzzi and Blakeslee, 2007). Swiss chard is a very good source of
vitamins C, A and B, phenolic acids (syringic, caffeic and p-coumaric), flavonoids (kaempferol,
quercetin and glycosides derived from apigenin) and minerals such as iron, potassium,
calcium, magnesium and manganese, which additionally contributes to the functionality of
Swiss chard (Ninfali and Angelino, 2013; Pyo et al., 2004). Swiss chard also contains a good
amount of dietary fibers and proteins, which also enhances its role in blood sugar regulation
(Kolota et al., 2010; Ninfali and Angelino, 2013; Pyo et al., 2004; Sacan and Yanardag, 2010).
Factors such as climate, environmental conditions, applications of different rates of
fertilizer, the time of harvesting, germination, plant physiology state, all affect nutritional
properties and phytochemical content of the food crops (Lombardo et al., 2017; Miceli and
Miceli, 2014). The application of the optimal amounts of nitrogen fertilizers is a common
farmer practice that aims to maximize the economic return and maintain environmental
quality (Miceli and Miceli, 2014). The treatment of Swiss chard with different amounts of
fertilizer impacted certain nutritive factors (Miceli and Miceli, 2014) but the data regarding
the effects of fertilization on phytochemical content of Swiss chard are scarce. The adequate
water supply is essential for crops’growth, as well as the adequate applications of fertilizers
(Mogren et al., 2016; Ertek and Kara, 2013). Reduced water supply has been used as one of
the stress methods of producing crops that contain a higher level of micronutrients such as
ascorbic acid (Mogren et al., 2016). It was shown that restricted irrigation could lead to
maintaining the post-harvest quality of spinach (Mogren et al., 2016).
Today, when we are facing global problems such as hunger and micronutrient
deficiency, especially in developing countries and coexisting obesity and related chronic
diseases in developed ones (Frison et al., 2006), the cultivation of Swiss chard should be
encouraged because it is an excellent source of nutrients and various phytochemicals, and at
the same time a cheap food crop (Ninfali and Angelino, 2013).
The aim of this study was to determine nutrient and phytochemical content of Swiss
chard of Montenegrin origin, as well as, to assess the effect of different levels of fertilization
and irrigation on nutritional and phytochemical composition of the Swiss chard.
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2. Material and methods
2.1 Plant material
Swiss chard (Beta vulgaris sub. cicla), Verca F1 hybrid (Clause, France) was grown in the
experimental field of Ljeskopolje (coordinates 42.447155°N and 19.206541°E), in Podgorica,
Montenegro. Seeds of chard were sown in plastic trays with cells supplied with substrate.
Seedlings were grown in a greenhouse, and those with four to five leaves were transplanted
on the field in mid-April, 2015. The plot size was 10.5 m
2
(3 m ×3.5 m). Chard is planted into
rows spaced 25 cm apart, the eight lent per experimental plot. The trial consisted of 3 control
plots and 27 plots which were treated with three different levels of fertilization and three
different norms of irrigation.
An experiment was conducted on a sandy clay loam soil with pH ¼5.5. The soil was
carbonate-free, medium level of humus (4.17 percent). On March 2015, for the purpose of
improving growing conditions, the soil was treated with Ca(OH)
2
-70 g/m
2
and organic pellet
fertilizer 163 g/m
2
(composition: organic matter 67 percent; total nitrogen 5 percent;
phosphorous (P
2
O
5
) 3 percent; potassium (K
2
O) 2 percent and pH ¼7.2) to give final
pH ¼5.7.
Fertilization (N, P, K) included the reduced nutrition with 50 percent of the crop
requirements (F50), optimal nutrition with 100 percent of the crop requirements (F100),
and the increased nutrition with 50 percent higher than the crop requirements (F150).
Irrigation included the reduced amount of applied water (representing 50 percent of the
calculated values of the optimal crop evapotranspiration, I50), the optimal amount of
applied water (I100) and the increased amount of applied water (representing 150 percent
of the calculated values of the optimal crop evapotranspiration, I150). The doses of
fertilizers were determined based on the literature data on the needs of the individual
cultures, the results of soil analysis, and the fertilizer manufacturer’s recommendations.
The total amounts of nutrients applied for optimal nutrition treatment (F100) were:
16.75 g N/m
2
, 12.36 g P
2
O
5
/m
2
and 14.79 g K
2
O/m
2
. The mature leaves of chard were
removed manually, and the experiment for this study started on June 6. During our trial,
NPK fertilizer was applied, on June 10, at rate −2.10 g N/m
2
,1.53gP
2
O
5
/m
2
and 1.57 g
K
2
O/m
2
( for F100 treatment).
The irrigation norms were calculated by using ACLIMAS-EXCEL-IRP program which is
based on modified FAO 56 Penman-Monteith method (Allen et al., 1998), recommended for
estimating the potential and reference evapotranspiration rates. The system for water
distribution consisted of suitable valves, water volume meters and polyethylene tube
(laterals) with inline dripper. During the period of experiment ( June 6–22) the sum of water
applied ten times was 0.059 m
3
/m
2
for optimal irrigation treatment (I100).
Data on climatic conditions from 6 to 22 June were collected from the agro-meteorological
station Davis Vantage Pro 2, installed at site Ljeskopolje, whereby the average temperature
in this period had the daily value (DV ) of 23.81±2.31°C (maximum average temperature was
26.4°C, while the minimum was 20°C). The sum of precipitation for growing period of chard
was 21.4 mm, reaching maximum sum per day to 11.4 mm on June 21.The experiment had a
completely randomized design with three replicates per variant.
Swiss chard samples were harvested on June 22, yield and leaf length were measured
in situ. These samples were transported on ice in plastic bags in a handheld fridge to
the laboratory of the Institute of Public Health of Montenegro where all of the samples
were analyzed.
2.2 Sample preparation
The edible parts of Swiss chard, leaves and steams were washed with tap water and double
distilled water. They were drained completely, dried over filter paper and homogenized with
domestic processor in a dark room at 25°C. The proximate analysis, individual mineral
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chard from
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components, vitamin C, chlorophyll a, chlorophyll b and carotenoid contents were
determined after the homogenization of fresh Swiss chard. The rest of the samples were
packaged in a vacuum plastic bag and stored at −26°C until analysis.
2.3 Proximate analysis of macronutrients
The moisture content was determined by drying the edible parts of Swiss chard in an oven at
105°C until constant weight was obtained. The samples were incinerated in a muffle furnace
at 520°C until constant weight was obtained in order to evaluate the ash content. Total lipids
were extracted by repeated washing (percolation) with petroleum ether in Soxhlet extraction
systems at 45–60°C. The total nitrogen content was evaluated using the Kjedahl method and
the protein content was calculated from the total nitrogen content multiplied by 6.25. The
above-mentioned macronutrient analyses were conducted in accordance with the AOAC
methods: 950.46, 930.30, 948.15, 991.20, respectively (Latimer, 2012). Crude fibers (raw
cellulose and lignin) were determined according to the Scharrer–Kurscher method (Matissek
and Steiner, 2006). Total carbohydrates were calculated by subtracting the total amount of
proteins, total lipids, total ash, moisture content and dietary indigestible fibers out of a
hundred. The energy (kJ) was calculated according to the formula (USDA, 2014):
E¼2:44 gproteinsðÞþ8:37 glipidsðÞþ3:57 g carbohydratesðÞ
½
=0:24:
2.4 Determination of the individual mineral content
The individual mineral profile was estimated according to AOAC method 984.15 (Latimer,
2012). The homogenized samples (1 g) were digested with 1 mL of 30 percent H
2
O
2
and
7.0 mL of HNO
3
by using microwave digestion method (ETOS1, Milestone). After the
digestion procedure and subsequent cooling, the digested samples were diluted with water
to a final volume of 25.0 mL and measured by inductively coupled plasma-optical emission
spectroscopy (ICP-OES, SPECTRO ARCOS, Kleve, Germany). The results were expressed in
mg or µg per 100 g fresh weight (FW).
2.5 Vitamin C determination
The content of vitamin C in Swiss chard samples was measured on a Reflectometer
RQflex10 Reflectoquant using Reflectoquant ascorbic acid test strips (Reflectoquant®
Ascorbic Acid Test, 2016). The results were presented as mg of vitamin C in 100 g FW.
2.6 Chlorophyll a, chlorophyll b and total carotenoid content
Chlorophyll a, chlorophyll b and total carotenoids content were determined according to the
procedure developed by Sumanta et al. (2014). The amount of 0.5 g of homogenized samples
was mixed with 10 mL of 80 percent acetone on vortex and then the mixture was
centrifuged. The supernatant was separated and 0.5 mL of it were mixed with 4.5 mL of
acetone. The absorbance of the final mixture was measured at 470, 663.2 and 646.8 nm in a
spectrophotometer (Varian, Cary 3E UV-VIS Spectrophotometer) and the equation used for
the quantification of chlorophyll a, chlorophyll b and carotenoids is given as follows:
Chlorophyll a Chl aðÞ¼12:25A663:2279A646:8;
Chlorophyll b Chl bðÞ¼21:5A646:85:1A663:2;
Carotenoids ¼1;000A4701:82Chl a85:02Chl bðÞ=198:
The results were expressed in mg per 100 g FW.
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2.7 Sample preparation for antioxidant analysis
The homogenized frozen samples were drained completely and then dried on a filter paper.
The homogenized samples (5 g) were weighted and dissolved in 50 mL of distilled water and
then heated for 45 min at 60–70°C. After the extraction and filtration, the water was
evaporated using Rotation Vacuum Evaporator. The resulting brown solid residue was
dissolved in distilled water to yield concentrations from 1 to 10 mg/mL of water extract.
These extracts were used for total phenol content, total flavonoid content (TFC) and for
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assessment.
2.8 Total phenol and total flavonoid content assessment
The total phenolic content (TPC) of the extract was determined with Folin–Ciocalteu (FC)
reagent, according to the Slinkard and Singleton (1977) method with some modifications.
The 0.1 mL of the Swiss chard extract (1 mg/mL), 4.5 mL of distilled water, 0.1 mL of FC
reagent (three times diluted) and 0.3 mL 2 percent Na
2
CO
3
solution were added and mixed
well. Absorbance of the mixture was read at 760 nm in a spectrophotometer, after 2 h. TPC
was calculated from the standard calibration curve obtained from gallic acid and the results
were expressed as µg of GAE per mg of the water extract.
According to the method of Sakanaka et al. (2005), the TFC of the chard extract was
determined using catechin as a standard flavonoid compound. A 0.5 mL of the chard extract
(1 mg/mL) was taken in a test tube and 2.50 mL of distilled water and 0.15 mL of a 5 percent
NaNO
2
solution were added. After 6 min, 0.3 mL of a 10 percent AlCl
3
solution was added
and allowed to stand at room temperature for 5 min and then 1 mL of 1M NaOH was added
to the test tube. The solution was then diluted with distilled water to make the final volume
up to 5 mL. The absorbance was read at 510 nm. TFC was calculated from a calibration
curve, and the result was expressed as mg of CAE per mg of the water extract.
The absorbances were measured using a spectrophotometer (Varian Cary 3E UV-VIS).
2.9 DPPH free radical scavenging activity
The antioxidant activity of the extract was determined by the DPPH assay according to the
procedure developed by Brand-Williams et al. (1995). A solution of DPPH in methanol
(6 ×10
−5
M) was prepared freshly. A 3.9 mL aliquot of this solution was mixed with 0.1 mL
of the samples at varying concentrations (1–10 mg/mL). The solutions in the test tubes were
shaken well and incubated in the dark for 30 min at room temperature. The decrease in
absorbance was measured at 517 nm against methanol. The percentage of the free radicals
inhibitions due to the antioxidant property of the extracts was calculated using the formula:
%¼100 Ablank–Asample
=Ablank
2.10 Statistical analysis
The results were expressed as a mean ±SD. Data were processed using IBM SPSS Statistic
v23 (Armonk, New York). The significant differences between the means, for variants with
different levels of fertilization and irrigation (without controls), were determined with
two-way ANOVA and Duncan’s test at po0.05.
3. Results and discussion
3.1 Yield and some plant characteristics
The growth parameters (yield and leaf length) of Swiss chard are presented in Table II.
Plant nutrition through application of NPK fertilizers can influence growth, plant
morphology, yield and quality (Ullah et al., 2016). Nitrogen is essential in many of the
process needed to carry out growth, it is also vital to chlorophyll and significant component
in amino acids, so it is required for virtually every physiological processes in plants;
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chard from
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Potassium helps regulate plant metabolism and Phosphorous is involved in the metabolic
process responsible for transferring energy from one point to another in the plant and it is
critical in flowering (Hopkins and Hüner, 2014). The results showed significant ( po0.05)
response for yield and leaf length (Table II), to different rates of NPK fertilizer application,
while there were no effects of irrigation and F×Iinteraction. For yield, and leaf length, F150
treatment recorded significantly higher values than F100 and F50 treatments (Table V ). No
significant difference in these parameters between F50 and F100 treatments was found. The
yield and chard’s leaf length showed positive responses to fertilizer treatments as expected
since N, P, K play a vital role in plant growth and production (Ullah et al., 2016). This is in
agreement with the findings of Miceli and Miceli (2014) who reported that the increasing
level of nitrogen increases yield and some of tested morphological characters of Swiss chard.
3.2 Proximate analysis of macronutrients
The macronutrients composition of differently treated Swiss chard samples is presented
in Table I. All the macronutrient parameters presented in Table I were not significantly
affected by fertilization, irrigation and F×Iinteraction ( pW0.05), except for the total
protein content which was affected by fertilization. It was observed that increasing rates
of a fertilizer increased the protein content(TableV).Themoisturecontentofthe
samples ranged between 87.95 and 90.21 g/100 g FW. Swiss chard samples in this study
contained less than 0.45 g of fat /100 g FW. The mean value of indigestible dietary fibers
was 0.77 g/100 g FW. Total carbohydrates were the second major chemical constituent
found in chard samples. Their content ranged from 4.65 g/100 g (I0F100) to 6.84 g/100 g
(I100F0) and these values are higher compared to 3.74 g/100 g FW found by the US
Department of Agriculture (USDA, 2014). The mean value of the ash content in tested
samples was 1.86 g/100 g. Energy values of the tested Swiss chard samples ranged from
105.37 to 136.75 kJ. The higher energy values compared to the values used by USDA
(2014) were a consequence of higher carbohydrate content. Additionally, due to the low
energy values as well as the low fat content Swiss chard could be recommended for
controlling cholesterol, prevention of obesity and weight reduction.
3.3 Individual mineral content
The individual mineral compositions of the treated samples are shown in Tables III and IV.
Different irrigation levels had no effect on mineral parameters ( pW0.05). The fertilization
affected only phosphorous content ( po0.05), in the sense that concentration of P for F150
treatment was significantly lower than for F100 treatment (Table V). Fertilization did not
affect the other tested mineral components ( pW0.05).
The interaction effect between fertilization and irrigation (F×I) was significant only for
Na and Cu contents ( po0.05) as it shown in Tables II and III, considering all tested
parameters. Namely, the content of Na and Cu in Swiss chard was at minimum for I100F150
treatment, but at maximum for I100F150 treatment. It can be explained by the opposite
effect of concentration of nutrients in soil solution in the conditions of increased and reduced
fertilization on sodium and copper uptake by Swiss chard under optimal water supply.
The major elements essential for normal human health are presented in Table III. From
among these macroelements, potassium was found to have the highest concentrations in
chard samples. The level of Na, K, Mg and Ca in chard samples ranged from 113.71 to
169.24, 339.04 to 417.19, 91.83 to 122.41 and 153.94 to 231.14 mg/100 g FW, respectively.
In comparison to USDA (2014) reported values, K mean concentration in our chard
samples (372.48 mg/100 g FW) was similar (379.00 mg/100 g FW), Mg and Na
concentrations (101.99 and 139.61 mg/100 g FW, respectively) were slightly higher
or lower (81.00 and 213.00 mg/100 g FW, respectively) and the concentration of Ca
(178.59 mg/100 g FW) was about four times higher (51.00 mg/100 g FW). A total of 30 g of
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Treatment Moisture Ash Protein Total lipids Crude fibers Total carbohydrates Energy (kJ) Yield (kg/parcel) Leaf lent (cm)
Controls
I0F0 89.47 ±0.82 1.91 ±0.06 1.93 ±0.13 0.45 ±0.04 0.62±0.11 5.62 ±0.63 136.75 ±0.25 3.35 20.9 ±1.38
I100F0 88.21±1.20 1.87 ±0.06 1.89 ±0.06 0.45 ±0.05 0.74 ±0.15 6.84 ±1.23 109.12 ±0.46 4.00 23.1 ±1.98
I0F100 90.21±0.60 1.80 ±0.11 2.41 ±0.09 0.37 ±0.04 0.56 ±0.06 4.65 ±0.60 118.50 ±0.38 5.45 22.2 ±2.19
Treatment
I50F50 88.38±0.94 1.87 ±0.13 2.26 ±0.10 0.41 ±0.03 0.73 ±0.12 6.35 ±1.19 133.21 ±3.17 8.53 27.20 ±0.95
I100F50 89.72 ±0.62 1.91 ±0.06 2.31 ±0.14 0.41 ±0.02 0.65±0.05 5.00 ±0.54 105.37 ±1.85 10.17 29.80 ±3.12
I150F50 89.06 ±0.97 1.91 ±0.05 2.33 ±0.14 0.38 ±0.06 0.67±0.25 5.65 ±0.98 120.96 ±3.23 9.47 29.30 ±1.01
I50F100 88.89 ±0.81 1.85 ±0.04 2.51 ±0.12 0.36 ±0.06 0.72±0.05 5.67 ±0.67 112.21 ±2.95 11.27 30.00 ±0.89
I100F100 88.44 ±1.30 1.81 ±0.08 2.35 ±0.06 0.38 ±0.08 0.77±0.35 6.25 ±1.29 131.50 ±4.25 10.28 28.97 ±1.60
I150F100 89.61 ±0.59 1.83 ±0.08 2.31 ±0.09 0.36 ±0.03 0.64±0.03 5.25 ±0.64 113.71 ±1.75 13.60 30.33 ±1.76
I50F150 87.95 ±1.40 1.92 ±0.09 2.75 ±0.05 0.31 ±0.03 0.76±0.07 6.31 ±1.33 133.67 ±5.04 14.41 33.00 ±0.36
I100F150 88.77 ±0.18 1.78 ±0.03 2.63 ±0.15 0.36 ±0.03 0.59±0.11 5.87 ±0.05 126.29 ±0.74 15.42 31.87 ±2.58
I150F150 88.20 ±0.81 1.88 ±0.08 2.63 ±0.14 0.38 ±0.04 0.68±0.06 6.23 ±0.93 131.83 ±3.14 15.30 33.70 ±1.82
ANOVA
Fertilization(F) 1.814 1.663 25.585* 1.918 1.148 0.532 –10.396* 13.013*
Irrigation(I) 1.133 1.129 1.444 0.984 0.502 0.611 –0.624 0.917
F×I1.186 1.097 1.266 0.431 0.357 1.146 –0.518 1.101
Notes: –, not considered. Data are expressed as mean ±SD derived from triple analysis of same treatment from a different parcel, while controls were mean ±SD of
triplicate taken from the same parcel (n¼3). The plot size was 10.5 m
2
(3 m ×3.5 m). Unit was expressed in g/100 g FW. *Significant for po0.05
Table I.
The macronutrient
content and growth
parameters of
differently treated
Swiss chard samples
and the results of
two-way ANOVA
(F-values) for
fertilization (F),
irrigation (I) and
interaction F×I
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chard from
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Treatment Na K Mg Ca P
Controls
I0F0 133.01 ±0.21 349.14 ±0.14 95.24 ±0.09 166.44 ±0.09 37.76 ±0,24
I100F0 169.24 ±0.19 391.15 ±0.21 122.41 ±0.13 231.14 ±0.04 25.76 ±0.51
I0F100 165.20 ±0.24 355.21 ±0.15 111.11 ±0.11 179.04 ±0.05 26.20 ±1.29
Treatment
I50F50 128.28 ±18.76ab 375.05 ±19.45 98.42 ±6.87 183.13 ±23.71 46.08 ±0.55
I100F50 163.38 ±21.48c 417.19 ±51.66 108.34 ±13.21 202.92 ±28.46 54.43 ±5.21
I150F50 138.05 ±4.94abc 370.29 ±18.61 100.97 ±7.67 172.25 ±17.82 53.81 ±4.18
I50F100 139.24 ±15.95abc 376.37 ±24.07 98.87 ±10.16 165.85 ±18.19 52.39 ±2.86
I100F100 130.19 ±3.59ab 366.19 ±36.78 91.83 ±8.91 168.90 ±9.68 60.52 ±18.84
I150F100 121.65 ±10.90ab 339.04 ±67.11 96.68 ±2.70 166.75 ±2.49 63.96 ±9.85
I50F150 146.38 ±28.41bc 386.72 ±14.25 106.04 ±4.52 180.70 ±3.13 44.61 ±6.37
I100F150 113.71 ±9.18a 359.35 ±17.99 94.32 ±7.09 153.94 ±21.37 46.04 ±5.05
I150F150 126.96 ±9.81ab 384.15 ±8.37 99.68 ±2.76 172.11 ±9.19 52.40 ±3.33
ANOVA
F2.257 1.433 2.115 3.320 4.580*
I0.821 0.639 0.194 0.323 2.936
F×I3.712* 1.321 1.603 2.017 0.253
Notes: Data are expressed as mean ±SD derived from triple analysis of same treatment from a different
parcel, while controls were mean ±SD of triplicate taken from the same parcel (n¼3). Unit was expressed in
mg/100 g FW. Means for Na (affected by interaction F×I) in a column not followed by the same letter are
significantly different for po0.05. *Significant for po0.05
Table II.
Mineral composition
(macroelements) of
differently treated
Swiss chard samples
expressed as mg/100 g
FW and results
(F-values) of two-way
ANOVA for
fertilization
(F), irrigation (I) and
interaction F×I
Treatment Cu
d
Zn
d
Cr
#
Mn
d
Fe
d
Co
#
Controls
I0F0 0.46 ±0.01 0.59 ±0.04 8.21 ±0.14 8.83 ±0.15 2.14 ±0.01 3.41 ±0.01
I100F0 0.54 ±0.01 0.84 ±0.08 5.63 ±0.22 9.07 ±0.24 2.00 ±0.01 3.02 ±0.13
I0F100 0.48 ±0.05 0.77 ±0.04 7.64 ±0.19 8.53 ±0.35 2.75 ±0.01 3.62 ±0.07
Treatment
I50F50 0.33 ±0.05ab 0.58 ±0.04 4.17 ±0.66 7.90 ±1.28 1.23 ±0.39 2.90 ±0.43
I100F50 0.41 ±0.06c 0.70 ±0.12 3.97 ±0.83 9.74 ±1.38 1.29 ±0.03 2.84 ±0.28
I150F50 0.34 ±0.03ab 0.59 ±0.05 5.29 ±1.50 7.98 ±0.33 1.45 ±0.45 3.15 ±0.25
I50F100 0.38 ±0.03bc 0.66 ±0.08 3.24 ±1.10 8.13 ±1.17 1.24 ±0.05 3.13 ±0.12
I100F100 0.33 ±0.02ab 0.62 ±0.09 4.26 ±0.78 7.92 ±1.30 1.23 ±0.08 3.13 ±0.30
I150F100 0.32 ±0.03ab 0.63 ±0.09 3.36 ±0.08 8.11 ±0.47 0.97 ±0.08 2.89 ±0.40
I50F150 0.35 ±0.02abc 0.69 ±0.08 3.29 ±0.50 8.35 ±0.92 1.16 ±0.17 3.21 ±0.22
I100F150 0.28 ±0.02a 0.61 ±0.06 3.32 ±0.42 6.94 ±1.24 0.98 ±0.14 2.88 ±0.41
I150F150 0.35 ±0.04abc 0.70 ±0.05 3.90 ±1.11 8.68 ±0.91 1.15 ±0.31 3.38 ±0.21
ANOVA
F1.793 0.607 3.381 0.736 2.495 0.898
I0.671 0.012 1.134 0.033 0.244 0.899
F×I4.555* 1.794 1.285 2.574 0.956 1.295
Notes: Data are expressed as mean ±SD derived from triple analysis of same treatment from a different
parcel, while controls were mean ±SD of triplicate taken from the same parcel (n¼3). Unit was expressed in
d
mg/100 g FW and
#
µg/100 g FW. Means for Cu (affected by interaction F×I) in a column not followed by the
same letter are significantly different for po0.05. *Significant for po0.05
Table III.
Mineral composition
of essential trace
elements in chard
samples and results of
two-way ANOVA
F-values) for
fertilization (F),
irrigation (I) and
interaction F×I
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fresh Swiss chard (usual serving size) provided 1.74, 3.19, 7.65 and 11.17 percent (mean
values of tested samples) of DV of Na, K, Mg and Ca, respectively. The referent values of
DV (for Americans four years of age and older) used for Na ¼2.40 g; K ¼3.50 g;
Mg ¼0.40 g and Ca ¼1.00 g were taken from US Food and Drug Administration (FDA,
2013). The amount of phosphorus ranged from 25.76 to 63.96 mg/100 g, as shown in
Table III. Considering the mean value for the chard samples, 30 g of fresh chard provides
1.41 percent of the DV for phosphorous (DV for P ¼1.00 g) ( FDA, 2013).
The concentrations of the trace elements that are considered to be essential for normal body
functions are presented in Table III. Trace elements have a crucial role in numerous enzyme
reactionsinwhichtheygenerallyparticipateascofactors (Fraga, 2005). Concentrations of Cu,
Zn, Fe and Mn in chard samples were found in milligrams per 100 g FW, while Co and Cr were
found in micrograms. The concentration of Mn in all treated samples was, notably higher than
the amount found by USDA (2014) (0.366 mg/100 g FW), and higher than in other GLVs such as
collards Mn ¼0.658 mg/100 g FW, spinach Mn ¼0.897 mg/100 g FW and kale Mn ¼0.659 mg/
100 g FW (USDA, 2014). This large amount of manganese that was observed in the tested chard
samples is probably due to the effect of a relatively low soil pH or of root exudates on its uptake.
The concentrations of Cu, Fe and Zn ranged from 0.28 to 0.54 mg/100 g FW for Cu, 0.98 to
2.75 mg/100 g FW for Fe and 0.58 to 0.84 mg/100 g FW for Zn. Considering the mean values of
Swiss chard samples for Cu, Fe and Zn (0.38, 1.47 and 0.66 mg/100 g FW, respectively) and their
DVs (2.00, 18.00 and 15.00 mg, respectively) (FDA, 2013), 30 g of fresh Swiss chard provided
about 5.70, 2.45 and 1.32 percent of the DV, respectively. The acceptable range of intake of
manganese for adults (over 19 years of age) is 1.00–10.00 mg/day (EFSA, 2006), hence the
obtained values fit within this range.
The concentration of Co and Cr in chard samples ranged from 2.84 to 3.62 µg/100 g FW
and 3.24 to 8.21 µg/100 g FW, respectively (Table III). Cobalt is a part of vitamin B12 and
human needs for cobalt are usually fulfilled through the intake of vitamin B12, while
chromium is important for the metabolism of fats and carbohydrates (Fraga, 2005).
The estimated recommended daily intake for cobalt through vitamin B12 is 2.40 µg/day for
the adults (EFSA, 2006). Based on the chromium content in healthy diets, it was established
that an adequate intake for the adults aged 19–50 years is 35.0 µg/day (EFSA, 2006).
3.4 Chlorophylls and total carotenoid content in Swiss chard
Total chlorophyll (chlorophyll a +chlorophyll b) content in Swiss chard ranged between 26.81
and 65.67 mg/100 g FW, as shown in Table V. In their study, Miceli and Miceli (2014) found a
total mean value of chlorophyll concentrations in Swiss chard to be 33.50 mg/100 g FW.
The obtained values of total chlorophyll in the present study are similar to the previously
reported value for Swiss chard, but 4–6 times lower than the values reported for some other
GLVs in Mediterranean countries, such as garden rocket (359.62 mg/100 g FW), wild rocket
(303.23 mg/100 g FW) and dandelion (248.25 mg/100 g FW) (Žnidarčičet al., 2011). The
following values for total chlorophyll content were reported for other commonly consumed
GLVs such as spinach, lettuce and nettles: 150.30 mg/100 g FW, 18.80 mg/100 g FW and
207.00 mg/100 g FW, respectively (Duma et al., 2014).
In our study the total carotenoid content of Swiss chard was in the range from 4.41 to
13.40 mg/100 g FW (Table IV ). These quantities are greater than those found by Reif et al.
(2013) where total content of carotenoids in different cultivars of Swiss chard was 3.70 to
9.60 mg/100 g of fresh matter, expressed as a sum of lutein and β-caroten, with the highest
concentration value found in the reddish Swiss chard cultivar. Total carotenoid content of
some leafy vegetables such as dandelion, garden rocket and wild rocket were 6.34, 8.24 and
7.18 mg/100 g, respectively (Žindarčic ref ). Vegetables from Brassicaceae family contain the
highest reported carotenoid content which ranged from 0.50 to 19.60 mg/100 g of fresh
matter, also expressed as a sum of lutein and β-carotene content (Reif et al., 2013).
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chard from
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The differences in total carotenoid concentrations among these/those studies could be
attributed to different plant species/cultivars used, cultivation conditions, etc. (Žindarcic
et al., 2011; Kopsell et al., 2004) because the content of carotenioids in plants is determined by
physiological, genetic as well as environmental factors (Kopsell et al., 2007).
Chlorophyll and carotenoids contents of plants are sensitive to the conditions of the
plant’s growth, such as nitrogen nutrition through a fertilization treatment (Miceli and
Miceli, 2014). In our present study, statistically significant differences among different
treatments of fertilization were found for chlorophyll a, chlorophyll b content (Table V ),
while for carotenoids this level was near the limit ( p¼0.05). Fertilization with the highest
rate of fertilizer had a positive effect on chlorophyll a and chlorophyll b content. The
significant difference was recorded between F150 and F100 treatment (Table V ). This result
indicates that higher NPK application increases chlorophylls synthesis in Swiss chard
which is expected since the major nutrients (N, P, K) used individually or in combination
Treatment
TPC µg GAE/mg
of extract
TFC µg CAE/mg
of extract
Vitamin C
mg/100 g
Chlorophyll a
mg/100 g
Chlorophyll b
mg/100 g
Carotenoids
mg/100 g
Controls
I0F0 151.65 ±0.35 13.89 ±0.57 26.11 ±1.65 20.60 ±2.05 8.80 ±1.92 6.21 ±2.03
I100F0 126.45 ±0.28 11.04 ±1.16 33.05 ±0.95 17.60 ±1.26 9.21 ±1.29 4.41 ±1.30
I0F100 148.05 ±0.42 13.89 ±0.38 26.10 ±0.65 42.80 ±1.14 19.80 ±1.18 11.40 ±1.25
Treatment
I50F50 145.35 ±2.13 11.67 ±2.20 25.87 ±2.20 30.87 ±7.76 13.93 ±3.19 9.73 ±9.28
I100F50 132.00 ±11.25 11.50 ±1.09 24.67 ±2.08 40.40 ±8.84 18.07 ±2.55 11.87 ±12.56
I150F50 130.60 ±1.80 11.09 ±0.96 27.0 ±1.00 33.60 ±10.4 16.01 ±4.57 10.87 ±2.86
I50F100 132.20 ±10.20 10.84 ±1.02 25.07 ±3.49 23.73 ±3.24 10.67 ±2.50 7.13 ±0.90
I100F100 138.75 ±9.40 12.05 ±1.04 25.27 ±2.19 30.40 ±6.69 13.33 ±2.08 9.33 ±1.68
I150F100 128.85 ±13.21 11.19 ±0.60 25.07 ±0.90 29.93 ±8.00 13.67±2.33 9.13 ±3.77
I50F150 139.50 ±13.51 12.15 ±1.33 29.67 ±2.08 39.13 ±13.61 17.39±5.55 12.01 ±4.52
I100F150 136.8 ±18.17 11.20 ±0.94 28.33 ±4.16 45.40 ±13.16 20.27±6.04 13.40 ±5.72
I150F150 152.85 ±19.32 12.38 ±0.95 29.67 ±0.58 28.20 ±10.09 17.87 ±5.15 12.67 ±1.79
ANOVA
F1.276 1.122 7.727* 4.378* 4.912* 3.309
I0.164 0.427 0.562 1.376 1.466 0.703
F×I1.433 1.696 0.250 0.129 0.138 0.029
Notes: Data are expressed as mean ±SD derived from triple analysis of same treatment from a different
parcel, while controls were mean ±SD of triplicate taken from the same parcel (n¼3). CAE, catechin
equivalent; GAE, gallic acid equivalent; TFC, total flavonoid content; TPC, total phenol content. *Significant
for po0.05
Table IV.
Phytochemical
components of tested
Swiss chard samples
and results (F-values)
of two-way ANOVA
for fertilization (F),
irrigation (I) and
interaction F×I
Treatment F50 F100 F150
P mg/100 g FW 51.45 ±5.25ab 58.96 ±11.90b 47.68 ±5.67a
Protein g/100 g FW 2.30 ±0.11a 2.39 ±0.12a 2.67 ±0.12b
Chlorophyll a mg/100 g FW 34.94 ±8.90ab 28.02 ±6.35a 41.37±11.14b
Chlorophyll b mg/100 g 16.00 ±3.54ab 12.58 ±2.48a 18.52 ±5.00b
Vitamin C mg/100 g FW 25.84 ±1.89a 25.13 ±2.11a 29.22 ±2.44b
Yield kg FW/parcel 9.39 ±1.66a 11.72 ±2.63a 15.04 ±3.00b
Leaf length cm 28.77 ±2.09a 29.77 ±1.41a 32.86 ±1.78b
Notes: Data are expressed as mean ±SD for the same fertilization treatment (n¼9). Means in a row not
followed by the same letter are significantly different for po0.05
Table V.
Parameters of Swiss
chard affected by
fertilization
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promote quality of plants (Ohshiro et al., 2016). Similarly, Miceli and Miceli (2014) reported
that Swiss chard gives higher chlorophyll content with increased nitrogen application. The
same trend was observed for spinach (Nemadodzi et al., 2017). Irrigation and F×I
interaction had no significant influence on chlorophyll and carotenoids contents ( pW0.05).
3.5 Vitamin C
Vitamin C is a water-soluble vitamin which has a very powerful antioxidant capacity.
Since humans cannot synthesize vitamin C, it must be provided from the diet
(Hillstrom et al., 2003). Two different working groups observed that fertilization increased
the vitamin C concentration (Miceli and Miceli, 2014; Hassan et al., 2012). Our present study
showed that only fertilization significantly affected vitamin C content ( po0.05) in the sense
that the vitamin C concentration was significantly higher in the F150 treatment compared to
F50 and F100 (Table V ). These results indicate that increased NPK level promotes ascorbic
acid biosynthesis in Swiss chard, which is in agreement with research where increased
N applications rates resulted in higher vitamin C content (Miceli and Miceli, 2014; Dzida and
Pitura, 2008). On the other hand, some previous reports found that excessive N fertilizer
rates, decrease the concentration of vitamin C (Rajasree and Pillai, 2012; Stefanelli, Goodwin
and Jones, 2010). These opposite observations could be the consequence of different plant
species, climate condition, fertilization and other agricultural practices used. Vitamin C
content in chard samples ranged between 24.67 mg/100 g FW and 33.05 mg/100 g FW
(Table IV). The mean concentration value of vitamin C in all tested samples (26.90 mg/100 g
FW) was similar to those in spinach (28.10 mg /100 g FW) and collards (35.30 mg /100 g FW)
(USDA, 2014), but considerably lower than in kale (120.00 mg /100 g FW) and broccoli (89.20
mg /100 g FW), which had the highest vitamin C content among GLVs (USDA, 2014). The
recommended daily intake of vitamin C is 60.00 mg, which means that serving of 30 g of
fresh Swiss chard used in our study would provide 12.33–16.52 percent of DV (FDA, 2013).
3.6 Total phenol content and total flavonoid content
The consumption of vegetables that are rich in phenols and flavonoids is associated
with preventions of diseases caused by oxidative stress (Slavin and Lloyd, 2012; Ballistreri
et al., 2013).
The total phenol and flavonoid contents are shown in Table IV. The TPC varied from
126.45 to 152.85 µg of GAE/mg of water extract. Sacan and Yanardag (2010) measured
TPC of water extract from dry Swiss chard, and they found 31.09 µg pyrocatechol
equivalent/mg extract. The difference in results may be due to the use of different
equivalent, where gallic acid and pyrocatehol have different reactivity with FC reagents
(Everette et al., 2010). The TFC ranged from 10.84 to 13.89 µgofCAE/mgofextract
(Table IV). The used equivalent was the same as the one used by Sacan and Yanardag
(2010), and the values for TFC were in agreement with this study (11.88 µgofCAE/mgof
extract) (Sacan and Yanardag, 2010). The Swiss chard produced in this study is a good
source of this phytonutrients and should be recommended in an everyday diet. However,
TPC and TFC in different cultivars of Swiss chard and other vegetables vary widely and
they are difficult to interpret or compare to our study, because many factors have been
showed to influence TPC and TFC (Fonseca Maciel et al., 2011; Rop et al., 2010; Everette
et al., 2010). The effect of irrigation, fertilization and interaction F×Ion TPC and TFC
have not been showed in the present study ( pW0.05). Those observed conditions might be
due to the fact that phenolic and flavonoid compounds are secondary metabolites of the
plants tissues and they have the ability to synthesize them. The soil might also contain
enough of the nutrients required for the synthesis of these compounds in chard
irrespective to presence or absence of the fertilizer treatment. Additionally, the polyphenol
content in plants is affected by numerous factors such as genetic determinants,
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chard from
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environmental conditions, agricultural practices, post-harvest handling, etc. (Rop et al.,
2010; Cheynier et al., 2013), and all of them should be taken into account to explain the
results and no general role actually can be drawn.
3.7 Free radical scavenging activity
DPPH test is a widely used method for the antioxidant activities because it is a relatively
short-term test. DPPH radical scavenging activities of samples are presented in Figure 1.
The different fertilization, as well as the irrigation level, did not significantly change DPPH
activities of the tested chard samples ( pW0.05).
DPPH activity is best presented using IC
50
value, which is defined as the concentration of
the sample needed to scavenge 50 percent of DPPH present in the test solution. The IC
50
values of the tested samples were between 2.93 and 4.44 mg/mL (Figure 1). In their study,
Sacan and Yanardag (2010) reported IC
50
values for Swiss chard extract of 23.85 µg/mL
showing over 100 times higher scavening activity of chard extract compared to our study.
For methanol extract of Swiss chard the highest activity was found at 500 µg/mL and it was
87.00 percent (Pyo et al., 2004), while in our study it was 85.90 percent at a concentration of
10 mg/mL of the water extract. The obtained results showed a lower antioxidant activity
compared to the literature cited. The reported free radical scavenging activities of broccoli
had IC
50
values of 0.44 mg/mL for ethanolic extract and 2.13 mg/mL for water extract
(Bidchol et al., 2011). Other GLV such as spinach showed IC
50
value of 7.4 mg/mL for
methanol/water extract (Bajpai et al., 2005). Those values are consistent with our results
which indicate that Swiss chard, produced under different fertilization and irrigation
regiments, has a good antioxidant potential.
4. Conclusion
Swiss chard grown in Montenegro is rich in macronutrient components –proteins, dietary
fibers and macroelements such as Ca and Mg. Swiss chard grown in Montenegro has low
energy value, E¼122.75 kJ (mean value)/100 g of fresh Swiss chard. Additionally, the low
energy values as well as the low fat content recommend Swiss chard for the use in
controlling cholesterol, obesity prevention and weight reduction. Montenegrin chard could
be considered as an inexpensive but good source of numerous phytonutrients –vitamin C,
chlorophylls, carotenoids phenolic and flavonoid compounds and essential minerals such
as –Fe, Zn, Mn. Moreover, tested Swiss chard showed high chlorophyll and total
carotenoid content. Swiss chard shows a good antioxidant potential (IC
50
values ranged
from 2.93 to 4.44 mg/mL of water extract). The present study has shown that irrigation did
not affect any of the tested parameters ( pW0.05), while interaction between fertilization
100
80
60
40
20
0
I50F50
(IC50= 3.77)
I100F50
(IC50= 4.14)
I150F50
(IC50= 3.89)
I50F100
(IC50= 3.89)
I100F100
(IC50= 4.19)
I150F100
(IC50= 3.95)
Different treatments of Swiss chard samples with IC50 values
DPPH free radical scavenging
activity (%)
I50F150
(IC50= 3.68)
I100F150
(IC50= 3.67)
I150F150
(IC50= 3.79)
I0F0
(IC50= 3.21)
I100F0
(IC50= 4.44)
I0F100
(IC50= 2.93)
1mgmL
–1 2.5mg mL–1 5mgmL
–1 7.5mg mL–1 10 mg mL–1
Figure 1.
DPPH (1,1-diphenyl-2-
picrylhydrazyl) free
radical scavenging
activity of different
treatments of Swiss
chard’s water extracts
and related IC50
values
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and irrigation affected only Na and Cu content. The effect of irrigation on tested
parameters is therefore likely to be specific to the crop (variety) under study, and it is
difficult to evaluate especially under the open filed conditions because of the random
effects of rain, wind and water holding capacity of soil. Fertilization significantly affected
the protein content in the sense that increasing the level of fertilization gave a higher
protein content. Fertilization with the highest fertilizer rate had a positive effect on
chlorophyll a and chlorophyll b content ( po0.05), with a significant difference between
F150 and F100 treatments. It was shown that fertilization affected vitamin C content
(po0.05)inthewaythatthetreatmentofSwisschard with F150 resulted in significantly
higher values for vitamin C concentration, compared to F50 and F100 treatments. Beside
this, the fertilization treatment has a positive effect on yield and plant length indicating
that fertilization is important factor for plant growth.
In summary, it could be said that Swiss chard from Montenegro may be used as an
accessible source of natural phytonutrient components and should be included in the daily
diet in order to maximize the health benefits and reduce the risk of diseases. The nutritive
quality of Swiss chard may be improved by ameliorating the conditions of the plant’s
growth and with good agricultural practice.
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Further reading
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INTL/en/product/Ascorbic-Acid-Test, MDA_CHEM-116981 (accessed November 20, 2016).
Corresponding author
Ljubica Ivanovićcan be contacted at: ljivanovic7@gmail.com
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